Thrombin is a key enzyme in the coagulation cascade. By thrombin mediated proteolytic digestion of fibrinogen into fibrin monomer, a cascade reaction leading to clot formation is started. Clot formation is the first step in wound healing. In addition thrombin is a chemo attractant to cells involved in wound healing, and, the fibrin network formed act as a scaffold for collagen-producing fibroblasts, increases phagocytosis, promotes angiogenesis and binds growth factors thus further supporting the healing process. The rate of clot formation is dependent on the concentration of thrombin and fibrinogen. Because of the important function in clot formation thrombin has been utilised in a number of products intended for haemostasis and/or as tissue sealants or “glues”, both as stand-alone products (i.e. Thrombin-JMI) or in combination with fibrin or other compounds (i.e. Tisseel, Hemaseel, Crosseal). The potential fields of use are numerous; skin grafting, neuro surgery, cardiac surgery, toracic surgery, vascular surgery, oncologic surgery, plastic surgery, opthalmologic surgery, orthopedic surgery, trauma surgery, head and neck surgery, gynecologic and urologic surgery, gastrointestinal surgery, dental surgery, drug delivery, tissue engineering and dental cavity haemostasis.
So far the thrombin in approved thrombin-containing products on the market is derived either from human or bovine plasma. Using plasma derived protein confers several disadvantages as limited availability and safety concerns such as risk for transmission of viruses and prions and the risk of triggering autoantibody formation (bovine products). Cases where antibody formation due to bovine thrombin exposure has lead to significant bleeding disorders are known.
In vivo thrombin is obtained from activation of prothrombin through the coagulation cascade. Activation through the coagulation cascade is dependent on the presence of a functional GLA-domain containing 8-10 glutamic residues converted to gamma-carboxyglutamate. In vitro, also incomplete gamma-carboxylated prothrombin can be converted to thrombin by the use of prothrombin activators such as ecarin. Ecarin, a snake venom derived from the Kenyan viper Echis carinatus is a procoagulant, a protease which cleaves human prothrombin between residues Arg320-Ile321 to generate meizothrombin. Further autocatalytic processing results in the formation of meizothrombin desF1 and then alpha-thrombin, which is the mature active form of thrombin.
An ideal commercial thrombin manufacturing process would use a recombinant thrombin precursor and a recombinant protease produced at high productivity without addition of animal-derived components. Further requirements would be robust performance, convenience and low cost.
A big obstacle for efficient recombinant human thrombin (rh-thrombin) has been to obtain high yields of prothrombin. Although extensive efforts have been spent, obtaining high yields of prothrombin under conditions suitable for production of biologicals has long remained a challenge. Yonemura et al. (J Biochem 135:577-582, 2004) have used recombinant GLA-domain-less prethrombin digested with recombinant ecarin to generate recombinant human thrombin. The productivity of prethrombin at process scale was 150-200 mg/L, which is a modest productivity for commercial scale production. Recombinant production of ecarin has also been described in WO 01/04146. In this publication generation of rh-thrombin is exemplified by conversion of recombinant prothrombin produced in COS cells by a recombinant ecarin produced from CHO cells. However, the exemplified methods are not suitable for large-scale production and animal-derived components are used.
Recombinant ecarin is produced as a prepro-protein that needs to be activated. Problems to efficiently activate the r-ecarin are described in both publications and the suggested activation procedures are far from optimal.
Thus there is a need for improved methods to obtain recombinant human thrombin. During our efforts to obtain improved productivity of gamma-carboxylated human prothrombin we made the surprising discovery that co-expression with gamma-glutamyl carboxylase (GGCX) vastly improved also the productivity of incompletely carboxylated prothrombin (see WO2005038019).
The present invention describes a process to efficiently produce human thrombin from recombinant prothrombin obtained by the expression method as described in WO2005038019. Recombinant carboxylated or incompletely carboxylated prothrombin combined with recombinant ecarin has not previously been used for manufacturing of recombinant thrombin. Further, the procedure for activating recombinant ecarin is new. The methods described would be suitable for large scale rh-thrombin manufacturing without the addition of animal-derived components.